Molecular detection and differentiation of canine hemoplasma infections using RFLP-PCR in dogs in southern Iran

Two hemoplasma species are known in dogs: Mycoplasma haemocanis (Mhc) and Candidatus Mycoplasma haematoparvum (CMhp). The aim of the present study was to develop a novel restriction fragment length polymorphism (RFLP)-PCR method based on the 16S rDNA gene, using endonuclease Hind II, for detection and differentiation of canine hemoplasmas. Also, analysis of risk factors, clinical features and hematologic changes of positive cases was performed in dogs living in the Shiraz area of Iran. Blood samples were collected from anemic (packed cell volume (PCV) <= 35; n = 26) and control dogs (PCV > 35; n = 27) and were examined for the presence of canine hemoplasmas, using RFLP-PCR and 16S rDNA Sanger sequencing. The presence of Mhc (4 out of 53 cases; 7.5%) and CMhp ( 3 out of 53 cases; 5.7%) was confirmed by RFLP analysis of the amplified 16S rDNA and sequencing. No association was found between hemoplasma infection and anemia, health status, age, breed, gender, type of housing or the presence of other dogs in this study. Only the platelet number in Mhc infected dogs was statistically higher compared to CMhp positive and hemoplasma negative dogs. The present report documents the occurrence of Mhc and CMhp in southern Iran, and these hemotropic Mycoplasma infections must be expected even in the absence of clinical symptoms or hematologic abnormalities in dogs. For the first time, it has been indicated that RFLP-PCR assay is able to successfully distinguish hemotropic Mycoplasma in dogs.